Proteins play a very vital role in humans and perform different body functions which are vital to life. Their presence and/or absence have been correlated with several diseases. Therefore, detection and quantification of each protein is very important in medicine including in early detection of diseases. Typically assays like ELISA or western blot assays are used for such detection. The conceptual novelty of both these assays which allow detection of very low quantities of proteins is in the use of catalytic signal amplification: a single analyte molecule recruits an enzyme able to generate a multitude of reporter molecules, which is ultimately determined by the turn-over number of the enzyme. One enzyme which is very routinely used for signal amplification is horseradish peroxidise (HRP), which in the presence of H2O2 converts a non-chemiluminiscent molecule into a chemiluminiscent molecule with several thousand turnovers, thus decreasing the detection limits by several folds. Both these assays have severe limitations including (i) storage and handling of antibody/enzymes (ii) and prohibitive cost of antibody conjugated HRP, horse radish peroxidase (HRP) and (iii) long duration of the assays. In spite of these limitations they are being used extensively in analytical proteomics.
Detection of low concentration of proteins and other bio-markers is extremely important for early diagnosis of lethal diseases. Extensive efforts are being made by research groups to develop suitable chemical and biological probes that permit detection of a particular protein at low concentration in a complex proteome by the naked eye. High throughput detection of biomarkers can therefore be achieved without the use of much sophisticated instrumentation (which is therefore easy and inexpensive). Typical probes consist of two domains: chemical or biological domain, which specifically binds to the protein of interest; and a reporter molecule attached to it, which produces an output signal (e.g. fluorophore which gives a fluorescence output). For direct assays, in which fluorescent dyes such as fluorescein or rhodamine are commonly used as reporter molecules, the strength of the output signal is determined by the magnitude of the binding constant between the probe and the protein, together with the extinction coefficient and fluorescence quantum yield of the reporter molecule. Typically, therefore, detection limits are increased by improving the fluorescence output of the reporter molecule.
As an alternative, ABPP has emerged as a key technology in the evolution of functional proteomics. ABPP relies on the design of active-site directed covalent probes to investigate specific families of enzymes in complex proteomes. The fundamental building blocks of ABPP are small-molecule probes that covalently label the active site of a given enzyme or enzymes. These inhibitor probes which get covalently bound to the enzyme of interest are attached to a reporter tag to facilitate target characterization. Examples of reporter tags include fluorophores, biotin, and latent analytical handles such as alkynes or azides, which can be modified by click chemistry methods to visualize protein targets post-labeling by gel electrophoresis as has been shown by Cravatt et. al in Chemistry & Biology, April 2004, volume 11, issue 4, pp 535-546. Typically, fluorescent probes like rhodamine is used as the reporter tag and this limits the detection limit for the enzyme of interest. Further expensive analytical tools such as the gel doc system are required for visualization.
Article titled “In-gel detection of biotin-protein conjugates with a green fluorescent streptavidin probe” by AE Sorenson et al. published in Anal. Methods, 2015, 7, pp 2087-2092 reports a simple and reliable electrophoretic method to determine the relative extent of biotinylation of macromolecules. The method relies on complex formation between a biotinylated macromolecule and a streptavidin probe resulting in an electrophoretic mobility shift of the complex detectable by SDS-PAGE.
Thus there is an unmet need in the art since detection of very low concentration of proteins using fluorescent dye labelling requires very expensive fluorescence gel scanning systems.
Another drawback of state of art reagents available till date is that very few afford the biochemist the freedom to conduct a simple visual detection of proteins across a large range of the analyte concentration with a low, preferably extremely low limit of detection.
Accordingly, to overcome the above listed drawbacks of the various agents available for protein detection and quantification, the present invention provides a small molecule peroxidase mimic biuret-Fe-TAML i.e. horseradish peroxidase mimic FeIII-TAML complex of ligand as a catalytic probe for in-gel visual detection and quantitative detection of proteins in activity based protein profiling.